This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The bottom-up proteomic data is collected in a data-dependent fashion where a full MS scan is followed by several MSn scans that interrogate precursor ions selected from MS and/or fragmentation products generated via MSn. Real-time selection of the precursor ions and their fragmentation parameters is predetermined by the end user before the run and updated by the Xcalibur software. We use the ThermoElectron Component Object Model (COM) library to directly interact with an LTQ mass spectrometer and control many of the data-acquisition options not available via Xcalibur. Some of the new options are real-time adjustment of the CID or PQD collision energy, data-dependent HPLC flow, improved exclusion list in MudPIT experiments, and real-time database search.